Luminescence Microscopy with Enhanced Resolution

ABSTRACT

The invention is directed to a resolution-enhanced luminescence microscopy method in which a sample is excited to the emission of luminescence radiation through irradiation by excitation radiation, and an image of the luminescing sample is acquired. A first partial volume of the sample is irradiated by a first laser radiation field of the excitation radiation, and a second partial volume of the sample is irradiated by a second laser radiation field of the excitation radiation. The first partial volume of the sample and the second partial volume of the sample overlap one another partially but not completely. Only the first laser radiation field is modulated with a first frequency, and luminescence radiation is detected from the first partial volume of the sample with modulation filtering so that luminescence radiation from the second partial volume of the sample is suppressed.

RELATED APPLICATIONS

The present application is a U.S. National Stage application ofInternational PCT Application No. PCT/EP2007/007882 filed on Sep. 10,2007, which claims benefit of German Application No. DE 10 2006 046369.2 filed on Sep. 29, 2006, the contents of each are incorporated byreference in their entirety.

FIELD OF THE INVENTION

The invention is directed to resolution-enhanced luminescence microscopyand particularly to a method in which a luminescing sample to beexamined is illuminated by excitation radiation and an image of thesample that has been excited to luminescence is obtained. The inventionis further directed to a microscope for resolution-enhanced luminescencemicroscopy of a sample, means for exciting luminescence which irradiatethe sample with excitation radiation, and means for acquiring an imageof the excited sample.

BACKGROUND OF THE INVENTION

Luminescence microscopy is a typical field of application of lightmicroscopy for examining biological samples. For this purpose, certaindyes (phosphors or fluorophores, as they are called) are used forspecific tagging of samples, e.g., of cell parts. As mentioned, thesample is illuminated by excitation radiation and the luminescent lightthat is excited in this way is acquired by suitable detectors. For thispurpose, the light microscope is usually provided with a dichroicbeamsplitter combined with blocking filters which split off thefluorescence radiation from the excitation radiation and enable separateobservation. This procedure makes it possible to display individual,differently colored cell parts in the light microscope. Naturally, morethan one part of a specimen may also be dyed simultaneously withdifferent dyes attaching themselves specifically to different structuresof the specimen. This process is known as multiple luminescence. Sampleswhich luminesce, per se, that is, without the addition of dye, can alsobe measured.

In the present context, and as a general rule, luminescence is used asan umbrella term for phosphorescence and fluorescence and embraces bothprocesses.

Further, it is known to use laser scanning microscopes (LSM) for theexamination of samples which shows only those planes situated in thefocal plane of the objective in a three-dimensionally illuminated imageby means of a confocal detection arrangement (called a confocal LSM inthis case) or a nonlinear sample interaction (called multiphotonmicroscopy). An optical section is acquired and the recording of aplurality of optical sections at different depths of the sample thenmakes it possible by means of a suitable data processing device togenerate a three-dimensional image of the sample which is composed ofthe different optical sections. Accordingly, laser scanning microscopyis suitable for examining thick specimens.

Naturally, a combination of luminescence microscopy and laser scanningmicroscopy in which a luminescing sample is imaged at different depthplanes by means of a LSM is also used.

In principle, the optical resolution of a light microscope and of a LSMis diffraction-limited by physical laws. Special illuminationconfigurations such as the 4Pi arrangement or arrangements with standingwave fields are known for optimal resolution within these limits. Inthis way, the resolution can be appreciably improved over a conventionalLSM particularly in axial direction. Further, the resolution can beincreased by up to a factor of 10 over a diffraction-limited confocalLSM by means of nonlinear depopulation processes.

In recent years, a number of such techniques have been proposed ordeveloped which allow optical microscopy, particularly with LSM, tooperate with a resolution beyond the conventional Abbe diffractionbarrier [see Y. Garini, B. J. Vermolen and I. T. Young, “From micro tonano: recent advances in high-resolution microscopy”, Curr. Opin.Biotechnol. 16, 3-12 (2005)]. In this connection, there is a basicdistinction between nearfield and farfield methods, the latter beingespecially relevant because of their applicability to three-dimensionalimaging in the field of biomedicine.

In conventional fluorescence microscopy with a given numerical aperture(NA) and excitation wavelength, the above-mentioned nonlinearrelationship between the intensity of the exciting light and that of theemitted light must be produced in order to break the Abbe barrier oftransmissible spatial frequencies in a significant way [see R.Heintzmann, T. M. Jovin and C. Cremer, “Saturated patterned excitationmicroscopy—a concept for optical resolution improvement”, JOSA A 19,1599-1609 (2002)]. This is achieved, for example, by means ofmultiphoton microscopy [see W. Denk, J. H. Strickler and W. W. Webb,“Two-photon fluorescence scanning microscopy, a concept for breaking thediffraction resolution limit”, Science 248, 73-76 (1990)].

Other approaches include the methods of ground state depletion (GSD)[see U.S. Pat. No. 5,866,911 or S. W. Hell and M. Kroug,“Ground-state-depletion fluorescence microscopy: a concept for breakingthe diffraction resolution limit”, Appl. Phys. B 60, 495-497 (1995)] orstimulated emission depletion (STED) by Hell et al. [see DE 4416558 C2S. W. Hell and J. Wichmann, “Breaking the diffraction resolution limitby stimulated emission; stimulated-emission-depletion fluorescencemicroscopy”, Opt. Lett. 19, 780-782 (1994); T. A. Klar, E. Engel and S.W. Hell, “Breaking Abbe's diffraction resolution limit in fluorescencemicroscopy with stimulated emission depletion beams of various shapes”,Phys. Rev. E 64, 066613 (2001); V. Westphal and S. W. Hell, “NanoscaleResolution in the focal plane of an optical microscope”, PRL 94, 143903(2005)]. The common principle is based on the use of a distribution ofexcitation intensity and of saturation intensity in the sample, each ofwhich is structured in such a way that the maximum of the formercoincides with an interference minimum of the latter. A saturatedexcitation of the triplet state (hereinafter: GSD) or a saturatedde-excitation of the fluorescing state (hereinafter: STED) makes itpossible to deliberately quench the fluorescence of molecules which arenot located in the immediate vicinity of the interference minimum. Theradiation then proceeds only from the interference minimum. Theup-conversion fluorescence depletion technique established by Iketaki etal. functions in a similar way [see T. Watanabe, Y. Iketaki, T. Omatsu,K. Yamamoto, M. Salkai and M. Fujii, “Two-point separation insuper-resolution fluorescence microscope based on up-conversionfluorescence depletion technique”, Opt. Exp. 24, 3271-3276 (2003)].

DE 19908883 A1 proposes a direct saturation of the fluorescencetransition as a nonlinear process. The enhanced resolution is based on aperiodically structured illumination of the sample so that there is atransfer of high object space frequencies in the range of the opticaltransfer function of the microscope. The transfer can be achievedthrough costly postprocessing of data by computer.

SUMMARY OF THE INVENTION

Therefore, it is an object of the invention to provide a luminescencemicroscopy method and a luminescence microscope by which enhancedresolution is achieved without resorting to a plurality of wavelengthsor costly postprocessing of data by computer.

This object is met according to the invention by a resolution-enhancedluminescence microscopy method in which a sample is excited to emissionof luminescence radiation by irradiation with excitation radiation andan image of the luminescing sample is acquired, wherein a first partialvolume of the sample is irradiated by a first laser radiation field ofthe excitation radiation and a second partial volume of the sample isirradiated by a second laser radiation field of the excitationradiation, wherein the first partial volume of the sample and the secondpartial volume of the sample overlap partially but not completely, onlythe first laser radiation field is modulated with a first frequency, andluminescence radiation is detected from the first partial volume of thesample with modulation filtering so that luminescence radiation from thesecond partial volume of the sample is suppressed.

The above-stated object is further met though a resolution-enhancedluminescence microscope with means for irradiating a sample withexcitation radiation for exciting the emission of luminescence radiationand means for acquiring images of the luminescing sample, wherein themeans for irradiating with excitation radiation have means forirradiating a first partial volume of the sample with a first laserradiation field and means for irradiating a second partial volume of thesample with a second laser radiation field, wherein the first partialvolume of the sample and the second partial volume of the sample overlapone another partially but not completely, the means for irradiating thesample with the first laser radiation field have a modulator whichmodulates the first laser radiation field with a first frequency, andthe means for acquiring images detect luminescence radiation from thefirst partial volume of the sample with modulation filtering so that theluminescence radiation from the second partial volume of the sample issuppressed by the filtering.

Like GSD or STED, the method according to the invention and thecorresponding arrangement are single-point techniques in whichresolution is enhanced beyond the resolution of the laser radiationfield irradiation by the nonlinear cooperation of at least two laserradiation fields. Similar to DE 19908883 A1, direct saturation of thefluorescence transition can be applied as a nonlinear process. Butsimultaneous occupation of the triplet state no longer necessarily hasnegative results. It is essential that the fluorescence generated by theexcitation laser and the fluorescence generated by the saturation laserare separated from one another by modulation marking (MMF: ModulationMarked Fluorescence) and suitable frequency-sensitive and/orphase-sensitive detection.

Accordingly, two laser radiation fields are radiated in according to theinvention for increasing resolution. One of these two laser radiationfields is modulated. This laser radiation field is referred tohereinafter as center beam, center radiation or center laser radiation.A second laser radiation field whose radiation is not linearly modulatedis radiated in so as to overlap this first laser radiation field but notcompletely cover it. This second laser radiation field will be referredto hereinafter as the side laser radiation or side laser beam. The twolaser radiation fields are preferably structured in such a way that themaximum of the center laser beam coincides with the interference minimumof the side laser beam. In principle, the resolution is improved overthe resolution at which the center laser beam and side laser beam arecoupled in.

The approach according to the invention is a further development of theknown GSD and STED methods. However, it has some advantages over thesemethods which will be described briefly:

GSD is based on a saturation of the triplet state and therefore requiresmolecules with a high rate of intersystem crossing. There is no suchlimitation in modulation-marked fluorescence because neither the sidelaser beam T_(1,0) excitation nor the side laser beam S_(1,0) excitationhas an effect on the signal generated by the center laser beam. In theformer case, there is no fluorescence, whereas in the latter case nomodulated fluorescence occurs.

A drawback of the GSD method is the relatively long pixel dwell timerequired during scanning for image generation. In the first place, thisis necessary to achieve the stationary equilibrium needed for tripletsaturation (approximately 10 μs). In the second place, an initialrelaxation of all molecules back into the ground state is requiredfollowing the detection of a point for detecting the adjacent point(again approximately 10 μs). According to the invention, a saturation ofthe triplet state is not necessary, so that shorter dwell times can beused whose bottom limits are basically determined by the periods of thecenter laser beam modulations.

The intensities required within the framework of the invention are lowerthan those in STED.

A substantial advantage of the invention is the flexibility in thechoice of dye. While the intersystem crossing required in GSD islimiting, the STED method requires molecules which allow the mostefficient possible de-excitation of the S_(1,0) state. By contrast, theinvention makes it possible to use almost any dye whose level diagramcorresponds approximately to that shown in FIG. 1. Optimization of thereaction rates (e.g., with respect to moderately longer fluorescencelifetimes) is advantageous, but does not present a fundamentallimitation of the method. It must be emphasized that the essentialmodification with respect to conventional techniques is to be found morein the type of excitation and detection than in the choice of the sampleto be examined (in stark contrast to DE 10325460 A1, for example).

In the STED experiments realized up to the present, two wavelengths arerequired, whereas the invention works with only one wavelength. It isnot necessary to use a plurality of dichroic beamsplitters. Accordingly,a relatively simple construction can be used.

In STED experiments, it generally makes sense to use intensive pulsedlasers because the population of the excited state should be decreasedin the side laser beam area before fluorescence tales place. Incontrast, the invention can work with cw lasers irradiatingsimultaneously. However, it is advantageous to delay the irradiation bythe center laser beam with respect to the side laser beam byapproximately 10 ns because an extensive depopulation of the groundstate has already taken place by then (see FIG. 7). In somecircumstances, it is also possible to realize the modulation in the formof a pulsed laser.

The modulation-marked fluorescence (MMF) according to the inventionmakes it possible to improve high-resolution optical imaging to an evengreater degree. It presents an alternative to the two single-pointmethods GSD and STED which are already known. As in these known methods,the method according to the invention also works with at least two laserradiation fields (center laser beam and side laser beam). However,whereas the aim in GSD and STED is to completely suppress thefluorescence in the side laser beam area, in MMF the center sample areaand the laterally excited sample area are distinguished, e.g., bymodulated center laser beam excitation followed by phase-sensitivedetection of the signal of interest, and can therefore be separated. Theuse of a modulation-frequency-sensitive detection, e.g., by means oflock-in technology, is a central aspect for this purpose. Markedfluorescence in the side laser beam area, i.e., excitation throughphotons of the center laser radiation field, can be avoided by means ofan unbalanced intensity ratio between the laser radiation fields and asaturated depopulation of the ground state. A substantial advantage ofMMF over the known methods of GSD and STED is the freedom of choosingthe fluorophor and the possibility of operating the center laser andside lasers at the same wavelength.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is described more fully by way of example in the followingwith reference to the drawings.

FIG. 1 shows, by way of example, an energy diagram of a dye molecule orof a sample which is used within the framework of the invention;

FIG. 2 shows a possible intensity distribution for a side beam and acenter beam in the method according to the invention and in the deviceaccording to the invention;

FIG. 3 shows, by way of example, a schematic for a device according tothe invention;

FIG. 4 shows the intensity ratio between the center beam and side beamfor different embodiment forms of the invention;

FIG. 5 shows the equilibrium population as a function of the radiatedlaser beam intensity for a dye that can be used, for example, within theframework of the invention;

FIG. 6 shows the equilibrium population of the ground state along anormalized coordinate during irradiation by the side laser beam in themethod according to the invention for three different possible peakintensities;

FIG. 7 shows a diagram similar to that shown in FIG. 6 for other dyeparameters; and

FIG. 8 shows the population of the ground states and excited states as afunction of the illumination period in a particular embodiment form ofthe invention.

DESCRIPTION OF THE INVENTION

The typical arrangement, known per se, of the lowest energy level for afluorescing dye molecule is shown schematically in FIG., 1. Usuallyphotons of energy hv excite the molecules from state S_(0,0)(approximate vibrational ground state in the lowest electronic state) toa vibration-excited vibronic state S_(1,v). Conversely, stimulatedemission is, of course, also possible. Starting from S_(1,v), a fastvibrational relaxation takes place in state S_(1,0) and subsequently, ascompeting processes, either fluorescence or the transition to thetriplet state T_(i,v) with subsequent phosphorescence.

The excitation is carried out, according to the invention, by at leasttwo different light fields which are arranged in the same way as theexcitation laser radiation field and the saturation laser radiationfield in the known GSD or STED method. The use of lasers seems sensiblebut generally does not represent a limitation of the method.

FIG. 2 shows possible Airy intensity distributions of the laserradiation fields along the normalized coordinate v=kr*N.A. (where NA isthe aperture, k is the wave number 2 π/λ, and r is the radial distancefrom the center). The fields are designated in the following as centerbeam and side beam. They can have the same wavelength.

FIG. 3 shows an embodiment form of the device which in this case isconstructed similar to a Mach-Zehnder. A beamsplitter 2 divides thelight into a center beam path 4 and a side beam path 3 after a lightsource 1. A unit for spatial beam shaping 5 is located in the side beampath 3. This unit can comprise, e.g., an annular aperture which isimaged on the sample 10. Other possibilities are described, for example,in T. A. Klar, E. Engel and S. W. Hell, “Breaking Abbe's diffractionresolution limit in fluorescence microscopy with stimulated emissiondepletion beams of various shapes”, Phys. Rev. E 64, 066613 (2001). Ofcourse, two separate beam sources can also be used.

A modulation unit 6 is provided for the center beam path 4, which is notsubjected to spatial beam shaping, and modulates this beam with afrequency f_(c). After overlapping, the two beams are focused in thesample 10 in a diffraction-limited manner. An objective 9 is used forthis purpose. In addition, the focus is displaced in two dimensions by ascan unit 8.

Consequently, the overlapping of the center beam and side beam shown inFIG. 2 takes place at different locations in the sample 10. Thefluorescence excited in this way is recorded by a detector 12, e.g., aconfocal detector, via the objective 9, scan unit 8 and a preferablydichroic beamsplitter 7. A control unit (not shown) controls theoperation of the device.

It is crucial that the fluorescence signal measured in this way can beassociated with the respective beam 3, 4 by taking into account themodulation, i.e., the fluorescence is marked correspondingly. This isachieved by making use of modulation effects. In the simplest case, theside beam, for example, is not modulated (f_(s)=0), while the intensityof the center beam varies sinusoidally with a frequency f_(c) typicallyfrom 1-100 MHz, for which purpose the modulation unit 6 is introduced inthe center beam path. The fluorescence signal generated by the centerbeam is then likewise modulated with the frequency f_(c). This effectcorresponds among others to that which is also applied in the phasemethod for measuring fluorescence lifetimes (see, e.g., M. J. Booth andT. Wilson, “Low-cost, frequency-domain, fluorescence lifetime confocalmicroscopy”, J. Microscopy 214, 36 (2004)). Alternatively, the side beamand center beam are each modulated with frequencies f_(s) and f_(c),where f_(s)≠f_(c).

The modulation frequency f_(c) can be optimized in accordance with thedye. When detection is carried out in a phase-sensitive manner by meansof a lock-in amplifier (13) at frequency f_(c) as is shown by way ofexample in FIG. 3, the fluorescence signal generated by the center beamis extracted. Molecules which, in contrast, are excited (also) by theside laser beam show a non-modulated fluorescence and therefore do notcontribute to the signal at the output 14. In order to furtherstrengthen this effect, a polarization-sensitive detection can also takeplace making use of the fluorescence polarization.

In an arrangement corresponding to FIGS. 2 and 3, a resolution in themolecular range can be achieved when it is ensured that the probabilitythat molecules located in a sample area in which the side laser has anintensity other than zero will be excited by the side laser beam is ashigh as possible. In this connection, it does not matter whether thestate excited by the side laser beam has singlet characteristics ortriplet characteristics. It is important only that the intensity of theradiation emitted by the molecules excited by the side laser beam is notmodulated with frequency f_(s) and is therefore suppressed by thelock-in method. The lock-in technique is, of course, only one example ofa phase-sensitive or frequency-sensitive detection method.

The above-mentioned condition can be met, for example, when the sidelaser beam 5 has an intensity of sufficient magnitude that a saturationof the fluorescence transition occurs in the sample 10. In case of atwo-level system S_(0,0)/_(1,v) as in FIG. 1, in which there are novibrational states or triplet states and the fluorescence is stimulatedor tales place spontaneously proceeding from S_(1,v) the populationN_(1,v)=1−N_(0,0) o of the S_(1,v) state in stationary equilibrium isexpressed by:

${N_{1,v} \propto \frac{N_{p,s}}{{2\; N_{p,s}} + \frac{k_{fluo}}{\sigma}}},$

where N_(p,s) is the photon flux (of the side laser beam 5) and σ is theabsorption cross section of the optical transfer. The intensity of thefluorescence radiation is proportional to N_(1,v) by which the nonlinearrelationship between the intensity of the exciting light and that of theemitted light which was mentioned above as necessary for high resolutioncan be directly verified. For very high photon fluxes, equal occupationof the states and, therefore, saturation is achieved. Further, when theintensity of the modulated center laser beam 4 is very much smallercompared to the side laser beam 5 (i.e., N_(p,s)>>N_(p,c)), theprobability of fluorescence excitation by the center laser beam 4differs substantially from zero only at the interference minimum. Thisstate of affairs is shown clearly in FIG. 4, where the ratio I_(c)/I_(s)(center beam intensity to side beam intensity) is plotted as a functionof the normalized coordinate v corresponding to the intensity curvesshown in FIG. 2. Three different ratios of the respective integralintensities are taken into account. It will be seen that there is only avery low probability of excitation by the center laser beam 5 with anintegral ratio of 0.01 (i.e., the side laser beam 4 is 100 timesstronger than the center laser beam 5) in the range of 1 v 1>1. In thiscase, localized molecules correspondingly show hardly any modulatedfluorescence and are consequently suppressed duringmodulation-frequency-sensitive detection. This mechanism accordinglyachieves an increase in resolution beyond the diffraction boundary.

In a further development, the vibration levels of the individualelectronic states shown in FIG. 1 are included in the overlapping. Thetriplet state will continue to be left out of consideration for the timebeing (k_(ISC)=0). In order to determine occupation of the individualstates during irradiation by the side laser beam 4, rate equations canbe solved for different laser intensities in a first approximation(leaving aside coherence terms). FIG. 5 shows the population of statesS_(0,0) and S_(1,0) (N_(0,0) and N_(1,0), respectively) as a function ofthe laser beam intensity. By way of example, an absorption cross sectionof s=10⁻¹⁶ cm⁻², a vibrational relaxation rate of k_(vib)=(10⁻¹² s)⁻¹and a fluorescence rate of k_(fluo)=(2*10⁻⁹ s)⁻¹ were recorded. Valueswere shown for the stationary equilibrium which is always achieved afteran illumination period of about 10 ns. The sum of all of the populationsis scaled to 1. It will be seen that the ground state for intensitiesgreater than 100 MW/cm² is almost completely depopulated. Further, thecurves shown in FIG. 6 can be derived from the N_(0,0) o curve shown inFIG. 5 and demonstrate the depopulation of the ground state generated bythe side laser beam as a function of coordinate v. An intensity profilecorresponding to FIG. 2 with three different peak intensity values(intensity at maximum: 2 MW/cm², 20 MW/cm², 200 MW/cm²) was assumed. Asaturation effect leading to a constriction of the ground statepopulation can be seen clearly at the interference minimum. Now, if, inaddition, the modulated center laser is radiated in at a low intensity,the modulated excitation is substantially limited to the range of v=0.In this case, there is an interplay between the depopulation effect andthe above-mentioned circumstances of the different excitationprobabilities (FIG. 4).

Naturally, the specific shape of the curves in FIG. 6 depends amongother things on the properties of the selected fluorophor or sample 10.The example above is based on a fluorescence lifetime of 2 ns. A moreefficient saturation (and, therefore, lower intensities) can be realizedby using dyes with longer lifetimes. FIG. 7 corresponds to FIG. 6 andassumes a lifetime of 10 ns. It can clearly be seen that a flattening ofthe population curve occurs already at 20 MW/cm².

In a real system, the condition of a vanishing intersystem crossing isgenerally not entirely met. In this connection, typical rates ofK_(ISC)=(10⁻⁶ s)⁻¹ and k_(Ph)=(2*10⁻⁶ s)⁻¹ are assumed such as thosedocumented for rhodamine 6G (see M. Heupel, “Fluoreszenzspeltroskopieals neue Messmethode zur höchstempfindlichen Untersuchung transienterZustände”, dissertation, Uni-Siegen (2001)). FIG. 8 shows how thepopulations of states S_(0,0), S_(1,0) and T_(1,0) (see FIG. 1) changewithin 1 μs under these conditions assuming an irradiation intensity of20 MW/cm². Roughly this illumination period is necessary in order todetect a modulation in the range of several tens of MHz. It will be seenthat the lowest excited singlet state and the triplet state are occupiedapproximately equally at the selected parameters during the time periodshown here. In stationary equilibrium, the occupation shifts in favor ofthe triplet state, whose population in this example is approximatelytwice as high. In this case, the ground state is extensivelydepopulated. The extent of the depopulation often depends less on theillumination period than on the laser intensity that is used. When usinga side laser beam profile as shown in FIG. 2, a saturation effectsimilar to that in FIG. 6 or 7 results again. Since the occupation ofthe triplet state is often linked with a photobleaching process bysinglet oxygen (see C. Eggeling, A. Volkmer and C. A. M. Seidel,“Molecular Photobleaching kinetics of Rhodamine 6G under the conditionsof one- and two-photon induced confocal fluorescence microscopy”,ChemPhysChem 6, 791-804 (2005)), a rather short exposure period seems tobe advantageous. However, it must be ensured that the modulations of thecenter laser beam 5 remain detectable, i.e., a sufficient quantity offluorescence cycles is required.

It should be mentioned that laser radiation field arrangements andmodulation schemes other than those described above are alsoconceivable. For example, the fluorescence of molecules can be detectedin the area of overlap between two laser radiation fields by applyinglock-in detection with the sum frequency or difference frequencyf_(s)+f_(c) or f_(s)−f_(c), respectively (in this case, the designationscenter field and side field may no longer apply under certaincircumstances). When two simple (partially overlapping) Airy profilesare selected, a resolution similar to that in point spreadautocorrelation function imaging can be achieved [see G. J. Brakenhoffand M. Müller, “Improved axial resolution by point spreadautocorrelation function imaging”, Opt. Lett. 21, 1721-1723 (1996)].

While the invention has been illustrated and described in connectionwith currently preferred embodiments shown and described in detail, itis not intended to be limited to the details shown since variousmodifications and structural changes may be made without departing inany way from the spirit of the present invention. The embodiments werechosen and described in order to best explain the principles of theinvention and practical application to thereby enable a person skilledin the art to best utilize the invention and various embodiments withvarious modifications as are suited to the particular use contemplated.

1. A method for resolution-enhanced luminescence microscopy comprisingexciting a sample to the emission of luminescence radiation throughirradiation by excitation radiation, and acquiring an image of theluminescing sample, wherein a first partial volume of said sample isirradiated by a first laser radiation field of the excitation radiation,and a second partial volume of said sample is irradiated by a secondlaser radiation field of the excitation radiation, wherein said firstpartial volume of said sample and said second partial volume of saidsample overlap one another partially but not completely, only the firstlaser radiation field is modulated with a first frequency, andluminescence radiation is detected from the first partial volume of thesample with modulation filtering so that luminescence radiation from thesecond partial volume of the sample is suppressed.
 2. The methodaccording to claim 1, wherein said second partial volume of said sampleis brought to luminescence saturation by said second laser radiationfield.
 3. The method according to claim 1, wherein the intensity of saidsecond laser radiation field is at least 50-times greater than that ofthe first laser radiation field.
 4. the method according to claim 1,wherein the intensity of said second laser radiation field is at least100-times greater than that of the first laser radiation field.
 5. Themethod according to claim 1, wherein at least said first laser radiationfield is focused on said sample in a diffraction-limited manner.
 6. Themethod according to claim 1, wherein said second laser radiation fieldis modulated with a second frequency that differs from the firstfrequency.
 7. The method according to claim 1, whereinmodulation-filtering detection is carried out by lock-in technique. 8.The method according to claim 1, wherein the first frequency is between1 MHz and 100 MHz.
 9. A resolution-enhanced luminescence microscopecomprising means for irradiating a sample with excitation radiation forexciting the emission of luminescence radiation, and means for acquiringimages of the luminescing sample, wherein the means for irradiating withexcitation radiation have means for irradiating a first partial volumeof the sample with a first laser radiation field and means forirradiating a second partial volume of the sample with a second laserradiation field, wherein the first partial volume of the sample and thesecond partial volume of the sample overlap one another partially butnot completely, said means for irradiating the sample with the firstlaser radiation field having a modulatory which modulates the firstlaser radiation field with a first frequency, and said means foracquiring images detect luminescence radiation from the first partialvolume of said sample with modulation filtering so that the luminescenceradiation from the second partial volume of the sample is suppressed bythe filtering.
 10. The microscope according to claim 9, wherein saidsecond partial volume of the sample is brought to luminescencesaturation by the means for irradiating with second laser radiationfield.
 11. The microscope according to claim 9, wherein the intensity ofthe second laser radiation field is at least 50-times greater than thatof the first laser radiation field.
 12. The microscope according toclaim 9, wherein the intensity of the second laser radiation field is atleast 100-times greater than that of the first laser radiation field.13. The microscope according to claim 9, further comprising optics whichfocus the laser radiation for the first laser radiation field on thesample in a diffraction-limited manner.
 14. The microscope according toaccording to claim 9, wherein a modulator is provided which modulatesthe second laser radiation field with a second frequency that differsfrom the first frequency.
 15. The microscope according to claim 9,wherein said means for acquiring images have a lock-in amplifier, thefirst frequency and the signals of a detector being fed to the lock-inamplifier.
 16. The microscope according to claim 9, wherein the firstfrequency is between 1 MHz and 100 MHz.